Assessing the quality of bee honey on the basis of melissopalynology as well as chemical analysis

Melissopalynological and chemical analysis of honey provide us useful and valuable information about the botanical and geographical origin of honey. The data in question is very important for authentication as well as for testing the quality of honey, so this is considered the main method in honey regulation here, we have used chemical analysis and melissopalynology to evaluate different honey samples from two main Iranian hubs of honey. Sampling was carried out on two important poles in Iran’s honey production, the central Alborz region, and the mountainous Zagros ecosystems in the years 2020 to 2021. Therefore, 52 samples from Alborz (Northern Iran), as well as 42 samples from the Zagrosian ecosystems (western Iran) belonging to different ecological habitats, were collected. In addition, samples were taken at 7 altitudes from 0 to 3500 m a.s.l. Furthermore, in this study, various chemical analyses such as the effect of antioxidant activity, the amount of total phenolic content, pH, and moisture content of honey samples were evaluated. Our results showed that all honey samples were classified as polyfloral honey. Based on our findings, 57 honey samples (61%) contained the standard amount of pollen. A total of 42 plant families and 55 genera were identified in the studied samples, with the highest presence of Asteraceae, Fabaceae, Rosaceae, Apocynaceae, and Apiaceae. Finally, an antioxidant activity ratio of 19% to 98%, total phenolic content from 0.08 to 0.51 ppm, pH from 1.90 to 5.21, and moisture content from 13% to 18.40%.

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region, as the flowering season and nectar production can vary for a species in different regions 48 (Zimmerman, 1988). With the developing the of the honey market through the world 49 identification of botanical origin as well as its authenticity has received a lot of attention in 50 international societies (Kaskonine & Venskutonis, 2010). Fake honey and poor-quality honey 51 are major problems in the global honey trade. Honey, which is composed, of many stable 52 elements as well as pollen grains, collected by bees along with nectar. Melissopalynology as an 53 effective method as well as key criteria has been widely used to determine the purity, 54 geographical and botanical origins of natural honey (Maurizo 1975, ; Alvarez-Suarez 2017). 55 Louveaux (1970,1978) first founded this method. However, several laboratory procedures have 56 been developed to study of pollen grains in honey latter (Bryant & Jones 2001). 57 Melissopalynology studies evaluated pollen grains in honey (  less than one third of the average rainfall in the world (860 mm) (Amiri and Eslamian, 2010).

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Most of our honey samples were collected from Alborz and Zagros sections, which have the 128 highest honey production in Iran, which includes more than half of Iran's provinces, and most of 129 the collected samples were close to forested and protected areas ( Figure 1).     distilled water, but this time the samples were centrifuged for 5 minutes. In our study, we 199 repeated this process 4 to 7 times, depending on the amount of sugar in the samples, so that the 200 sugar in the honey was completely removed. But after the last centrifuge, we poured the final 201 remaining liquid into the watch glass and placed it on the hot plate so that the water evaporated 202 completely and the pollen grains remained at the bottom of the container. During the process, 203 great care must be taken to prevent any contamination from external pollen, which is from 204 previous honey products (use of disposable appliances is recommended as much as possible) or 205 pollen grains in the air (close windows and limit exposure). 206 We placed the prepared pollen grains on special bases and after covering the samples with gold 207 metal, we placed them inside the housing of HITACHI SU3500 scanning electron microscope 208 (SEM) machine made in Japan, and depending on the size of the sample, using this microscope, 209 it is possible to shoot surfaces with a magnification of 10 to 500,000 times and with a resolution Using scanning electron microscopy images, pollen grains obtained from honey sediment were 215 counted, counting at least 300 pollen grains is necessary to estimate the relative abundance of 216 pollen types and 500 to 1000 pollen grains are necessary to determine the relative abundance ( 217 Behm et al. 1996). We also used pollen atlases and websites to identify pollen, identification of   In this part of the study, some chemical analysis of honey such as antioxidant , total phenol, pH         The total phenol content of honey samples was between 0.08 to 0.59 micrograms. Differences in 278 phenolic content in honey samples may be due to different geographical origins or other factors 279 such as collection season and storage conditions. Also in this section, the pH of each honey 280 sample was examined.

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The lowest pH was 1.9 and the highest pH was 5.2. In the information obtained from measuring 282 the moisture content of each honey sample, it was determined that the maximum moisture 283 content was 18.8% and the minimum was 13%. We compared the humidity of honey samples 284 with the humidity of the areas from which the samples were collected, which shows that the

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In this study, 94 samples of honey collected belong to the main honey producing regions in Iran.

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In the results of this study, 42 plant families and 57 genera of these families were identified. Due   3) helps wound healing (Efem, 1988; 373 Molan, 1999), which is done by reducing the high alkaline environment in non healing wounds 374 to a more acidic healing environment. The global standard for pH of honey is 3.9 (3.2 -4.5).

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Based on the results of our studies, honey samples showed a pH 1.90 -5.21 and 48% of the total 376 samples are in the pH range of 2.5 -3.5 ( Figure 11).     concern that biodiversity loss is directly affecting ecosystem performance.

45
Honey is a precious natural product processed by bees from the nectar of flowers, and is stored in 46 the hive. Honey production in an area depends mainly on the vegetation and climate of thate 47 region, as the flowering season and nectar production can vary for a species in different regions 48 (Zimmerman, 1988). With the developing the of the honey market through the world     distilled water, but this time the samples were centrifuged for 5 minutes. In our study, we 199 repeated this process 4 to 7 times, depending on the amount of sugar in the samples, so that the 200 sugar in the honey was completely removed. But after the last centrifuge, we poured the final 201 remaining liquid into the watch glass and placed it on the hot plate so that the water evaporated 202 completely and the pollen grains remained at the bottom of the container. During the process, 203 great care must be taken to prevent any contamination from external pollen, which is from 204 previous honey products (use of disposable appliances is recommended as much as possible) or 205 pollen grains in the air (close windows and limit exposure). 206 We placed the prepared pollen grains on special bases and after covering the samples with gold 207 metal, we placed them inside the housing of HITACHI SU3500 scanning electron microscope 208 (SEM) machine made in Japan, and depending on the size of the sample, using this microscope, 209 it is possible to shoot surfaces with a magnification of 10 to 500,000 times and with a resolution      (Table 1) were identified.  The total phenol content of honey samples was between 0.08 to 0.59 micrograms. Differences in 278 phenolic content in honey samples may be due to different geographical origins or other factors 279 such as collection season and storage conditions. Also in this section, the pH of each honey 280 sample was examined.

281
The lowest pH was 1.9 and the highest pH was 5.2. In the information obtained from measuring 282 the moisture content of each honey sample, it was determined that the maximum moisture 283 content was 18.8% and the minimum was 13%. We compared the humidity of honey samples 284 with the humidity of the areas from which the samples were collected, which shows that the

314
In this study, 94 samples of honey collected belong to the main honey producing regions in Iran.

315
In the results of this study, 42 plant families and 57 genera of these families were identified. Due

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The amount of phenolic compounds is influenced by various factors such as plant type, 366 geographical origin and climatic conditions of the honey production site (Escuredo et al. 2012).

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Determining the amount of phenolic compounds in honey is a suitable factor to evaluate the 368 quality and therapeutic potential of honey (Ouchemoukh et al. 2007). In the results of our 369 analyzes, the samples showed phenolic compounds of 0.08-0.51 μg. Honey is recognized as a 370 23 treatment option for the treatment of acute and chronic wounds, and a wide range of in vitro and 371 in vivo research studies have been performed in this field (Subrahmanyam, 1998;Cooper et al. 372 2002; Johnson et al. 2005 ). Low pH of honey (0.4 -5.3) helps wound healing (Efem, 1988; 373 Molan, 1999), which is done by reducing the high alkaline environment in non healing wounds 374 to a more acidic healing environment. The global standard for pH of honey is 3.9 (3.2 -4.5).

375
Based on the results of our studies, honey samples showed a pH 1.90 -5.21 and 48% of the total 376 samples are in the pH range of 2.5 -3.5 ( Figure 11).  In this study, all honey samples showed a low moisture content (13% -18.80%), which 385 corresponded to the values (<20 <) set by the Codex standard for honey (Almentarius Codex, 386 1987). The parameter of quality and uniqueness of honey has been studied by various 387 researchers, this quality and credibility of honey is important for both buyers and producers.